The Journal of Neuroscience, November 3, 2004 • 24(44):9811–9825 • 9811 Behavioral/Systems/Cognitive Entorhinal Cortex Lesions Disrupt the Relational Organization of Memory in Monkeys Cindy A. Buckmaster,1,3 Howard Eichenbaum,4 David G. Amaral,5 Wendy A. Suzuki,6 and Peter R. Rapp1,2 1Fishberg Department of Neuroscience and 2Kastor Neurobiology of Aging Laboratories, Department of Geriatrics and Adult Development, Mount Sinai School of Medicine, New York, New York 10029-6574, 3Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11794, 4Laboratory of Cognitive Neuroscience, Boston University, Boston, Massachusetts 02215, 5The Medical Investigation of Neurodevelopmental Disorders Institute, University of California, Davis, California 95817, and 6Center for Neural Science, New York University, New York, New York 10003 Recent accounts suggest that the hippocampal system critically supports two central characteristics of episodic memory: the ability to establish and maintain representations for the salient relationships between experienced events (relational representation) and the capacity to flexibly manipulate memory (flexible memory expression). To test this proposal in monkeys, intact controls and subjects with bilateral aspiration lesions of the entorhinal cortex were trained postoperatively on two standard memory tasks, delayed nonmatching- to-sample (DNMS) and two-choice object discrimination (OD) learning, and three procedures intended to emphasize relational repre- sentation and flexible memory expression: a paired associate (PA) task, a transitive inference (TI) test of learning and memory for hierarchical stimulus relationships, and a spatial delayed recognition span (SDRS) procedure. The latter assessments each included critical “probe” tests that asked monkeys to evaluate the relationships among previously learned stimuli presented in novel combina- tions. Subjects with entorhinal cortex lesions scored as accurately as controls on all phases of DNMS and OD, procedures that can be solved on the basis of memory for individual stimuli. In contrast, experimental monkeys displayed deficits relative to controls on all phases of the PA, TI, and SDRS tasks that emphasized the flexible manipulation of memory for the relationships between familiar items. Together, the findings support the conclusion that the primate hippocampal system critically enables the relational organization of declarative memory. Key words: memory; hippocampus; relational representation; medial temporal lobe; nonhuman primate Introduction the underlying processing operations of the system rather than Normal memory depends on the hippocampal formation (widely the content of acquired information (Cohen and Squire, 1980; viewed on grounds of connectivity as comprising the dentate Graf and Schacter, 1985; Squire, 1992; Bechara et al., 1995; gyrus, hippocampus proper, subicular complex, and entorhinal Vargha-Khadem et al., 1997; Chun and Phelps, 1999). Specific cortex) (Insausti et al., 1987; Amaral and Witter, 1995) and asso- formulations include the proposal that the hippocampal forma- ciated perirhinal and parahippocampal cortices (Milner, 1972; tion subserves a broad organizational function, establishing rep- Zola-Morgan and Squire, 1993; Mishkin et al., 1997). The specific resentations of the relevant relationships between experienced cognitive operations implemented by this system in support of events and enabling the flexible utilization of memory (Eichen- memory, however, remain undefined. A popular view is that the baum et al., 1999; Eichenbaum, 2000). hippocampus is specialized or disproportionately devoted to The relational account receives support from human and ex- learning and memory for spatial information (Parkinson et al., perimental animal research. In one illuminating investigation, 1988; Nadel, 1991; Gagliardo et al., 1999; Matsumura et al., 1999; amnesics improved as quickly as controls on a probabilistic clas- Ekstrom et al., 2003). Other conceptualizations suggest that me- sification test of implicit learning that involved the nonconscious, dial temporal lobe memory is more accurately characterized by incremental acquisition of response biases. Unlike intact sub- jects, however, patients were unable to use memory flexibly and apply task knowledge under modified testing conditions (Reber Received April 22, 2004; revised Sept. 1, 2004; accepted Sept. 1, 2004. et al., 1996). Conceptually related impairments have been re- This work was supported by National Institutes of Health Grants NS32892, NS16980, and MH62448. We thank ported in mice and rats with hippocampal damage (Kogan et al., TracyAiello,JanineBeyer,PerikaDeroche,JeffreyDusek,PatrickHof,BentleyStrockbine,BrianLeonard,andHaydee Verscesi for expert collaborative and technical support and Andrew Leonard for graphics support. 2000; Agster et al., 2002; Fortin et al., 2002), including deficits in CorrespondenceshouldbeaddressedtoPeterR.Rapp,FishbergDepartmentofNeuroscience,MountSinaiSchool the ability to make inferential judgments about the relationships of Medicine, Box 1065, One Gustave L. Levy Place, New York, NY 10029-6574. E-mail: [email protected]. between familiar items presented in novel combinations and C. A. Buckmaster’s present address: Division of Veterinary Resources, Building 14A, National Institutes of Health, poor performance on previously acquired discriminations when Bethesda, MD 20892. DOI:10.1523/JNEUROSCI.1532-04.2004 items are presented in reverse of their original training order Copyright © 2004 Society for Neuroscience 0270-6474/04/249811-15$15.00/0 (Bunsey and Eichenbaum, 1996; Dusek and Eichenbaum, 1997). 9812 • J. Neurosci., November 3, 2004 • 24(44):9811–9825 Buckmaster et al. • Relational Memory in Monkeys Together, these findings suggest that relational information pro- sure, body temperature, and blood gases were monitored throughout the cessing may be a fundamental operating characteristic of mem- procedure. ory mediated by the mammalian hippocampal system (Eichen- To reduce bleeding, lidocaine (1%) with epinephrine (1:100,000) was baum et al., 1999). injected along the midline of the scalp before the first surgical incision. The present study was designed as a prospective test of the The skin and galea were then cut and reflected anteriorly and posteriorly, exposing the zygomatic arches. The zygomatic arches were removed, relational memory account in the nonhuman primate. Memory providing access to the ventrolateral aspect of the skull, and the temporal was evaluated in monkeys with bilateral aspiration lesions of the muscles were cut parasagittal to the anteroposterior midline, leaving a entorhinal cortex using established object recognition and dis- narrow strip of muscle attached to each side of the fascia for reattachment crimination tasks and a series of other assessments intended to at the end of the procedure. The muscle was gently retracted and ϳ2 ϫ 3 emphasize relational information processing. The entorhinal cm craniotomies were produced to expose the anterior and ventrolateral cortex is a major relay in the bidirectional exchange of informa- portions of the temporal lobe. Mannitol (30 ml at 25%, i.v., over 15 min) tion between the hippocampus and neocortex (Van Hoesen and was then provided to prevent edema and reduce brain volume in prepa- Pandya, 1975a,b; Van Hoesen et al., 1975; Insausti et al., 1987; ration for the entorhinal ablations. ϳ Witter et al., 1989; Suzuki and Amaral, 1994a,b), and damage in After rotating the animal 60° from supine position to permit optimal this region would be expected to substantially disrupt hippocam- access to the medial temporal lobe, the dura mater was cut and reflected, exposing the brain. The target area, manipulated into view under a sur- pal information processing. Previous studies, however, reported gical microscope, was bounded laterally by the rhinal sulcus and medially that entorhinal cortex lesions cause only mild and transient def- by the sulcus semiannularis (rostrally) and the choroidal fissure (cau- icits on standard memory tasks designed for monkeys (Meunier dally). Lesions were intended to spare the underlying white matter and et al., 1993; Leonard et al., 1995), perhaps supported by spared ablate all cortical tissue extending rostrodorsally into the temporal pole connectivity between the hippocampus and the perirhinal and and caudally to a point ϳ1 mm beyond the caudal limit of the rhinal parahippocampal cortices. Thus, we reasoned that against this sulcus. The surface of the entorhinal cortex was cauterized, and the dam- background of preserved function, lesions of the entorhinal cor- aged tissue was aspirated using a glass pipette with an angled tip. After tex would provide a strong experimental setting for testing the ablation, the dura mater was sutured and the craniotomy was packed proposal that relational representation and flexible memory ex- with Gelfoam (Upjohn, Kalamazoo, MI). The same procedures were pression critically require the integrity of the hippocampal used to make the contralateral lesion, and the wound was closed in ana- tomical layers. Once awake, the animal was allowed to recover in a warm- system. ing chamber and monitored closely. Cefazolin (50 mg, i.m.), dexameth- asone phosphate (1–2 mg, i.m.) or flunixin meglumine (Banamine, 4 mg, Materials and Methods i.m.), and buprenorphine hydrochloride (Buprenex, 0.15 mg, i.m.) were Subjects administered postoperatively to prevent infection, swelling, and pain,